The present invention relates to a method for the large scale production of preparation of mature and immature pancreatic endocrine cells and their use for treatment of diabetes mellitus.
Diabetes mellitus is defined as a chronic state of hyperglycaemia. This metabolic disturbance appears when insulin release has become insufficient, either as a result of a primary defect at the level of the insulin-producing beta cells or, secondary, when the beta cells fail to compensate for an increased peripheral resistance to insulin. The shift to elevated glucose levels can be counteracted by sustained adjustments in life style and by daily administration of hypoglycemic agents, under form of insulin injections or sulphonylurea tablets. Current treatment does however not succeed in a complete normalization of glucose homeostasis. Diabetic patients thus face the risk of developing chronic complications as a consequence of recurrent episodes of hyperglycemia. They are known to exhibit, as a group, a higher incidence of retinopathy and blindness, of nephropathy and renal failure, of neuropathy and amputations, of vasculopathy and cardiovascular disease. Diabetes is therefore considered as a major health problem. The disease is diagnosed in more than 5 percent of the Western population. Its impact on each patient""s quality of life is variable but life-long (Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, Diabetes Care 20,1183-1197, 1997).
A variety of stategies are currently explored in an attempt to find ways that stop the progression of the disease at any of its preclinical or clinical stages. Several are directed towards the pancreatic beta cells with the purpose of) reinstalling a functional beta cell mass that is sufficient to restore, at least in part, an endogenous control circuit in which insulin is released as a function of the metabolic needs. There are essentially two ways to achieve this goal. The first involves an implantation of foreign beta cells in order to replace the endogenous beta cell population or supplement it. It has been shown to correct the diabetic state in patients who had completely lost their endogenous beta cell mass (Warnock et al, Diabetologia 35:89-95, 1992; Ricordi et al, Transplantation 53;407-414, 1992; Gores et al, Lancet 341:19-21, 1993; Scharp et al, Transplantation 51:76-85, 1991). The second consists in administering drugs which increase the endogenous functional beta cell mass, either by inducing neoformation of beta cells, prolonging their survival or correcting their homeostatic function. Both strategies require the availability of large numbers of beta cells, either as cell grafts for implantation or as test model for screening and developing new drugs in the laboratory.
The number of patients who could benefit from a beta cell graft is, conservatively, estimated at 0.5 percent of the total population, which largely exceeds the number of candidates for other types of grafts. It is also clear that development of drugs acting on the beta cells involves extensive preclinical screening and testing for which large numbers of normal cells will be necessary. There is not yet a source of beta cells which can adequately fulfil both needs. Human pancreata have been used to produce beta cell preparations for transplantations as well as for in vitro studies but the number of donor organs is largely insufficient; moreover, criteria on human organ donation impede their use for drug development. These restrictions raise the need for producing beta cells from other species.
Among the larger mammals, pigs are considered as a potentially useful source of beta cell preparations since their use for medical applications faces fewer ethical obstacles than primates or other domestic animals, since pigs are relatively easy to breed and since porcine insulin is very similar to human insulin. Methods have been developed to isolate islet and tissue preparations from fetal, neonatal and adult pig pancreata. These preparations can normalize a diabetic state in immune-incompetent and in immune-competent mice (Korsgren et al, Surgery 113, 205-214, 1993; Korbutt et al, J Clin Invest 97, 2119-2129, 1996; Thomas et al, Transplantation 67:846-854, 1999; Lu et al, Xenotransplantation 5, 154-163, 1998). Fetal pig islet preparations have already been transplanted in diabetic patients, however without success (Groth et al, Lancet 344, 1402-1404, 1994). It is still unknown whether and if so, how successful xenotransplantation can be carried out in man. Use of reaggregated beta cell preparations with selected size and cellular composition might help a search for such conditions. Our studies in rodents have shown that purified islet endocrine cell aggregates exhibit a lower immunogenicity as allograft than intact islet tissue (Pipeleers et al, Diabetes 40, 908-919, 1991; Pipeleers et al, Diabetes 40, 920-930, 1991; Pipeleers-Marichal et al, Diabetes 40, 931-938, 1991, Pipeleers et al, Diabetologia 34,390-396, 1991). They also illustrate how variations in cellular composition influence the metabolic capacity of the grafts (Keymeulen et al, Diabetologia 40:1152-1158, 1997; Keymeulen et al, Diabetes 45,1814-1821, 1996). While these experiments demonstrated the usefulness of composing beta cell grafts in the laboratory, they did not offer an adequate methodology for clinical implantation. The methods that we used for composing the rat beta cell grafts do not allow large scale preparations of pancreatic endocrine cells. They involve prior isolation of the islets of Langerhans (herein defined as micro-organs with a diameter  greater than 100 xcexcm containing a mixture of endocrine cell types including the insulin producing beta cells) and thus discard beta cells that are present as single cells or as small cell aggregates; the single beta cells are assumed to be adjacent to immature endocrine cells, i.e. cells which can differentiate into beta cells. As a result of this removal, little is known about these beta cells and the immature endocrine cells. Their relative proportion (with respect to the numbers incorporated in islets) is however not insignificant during early phases of life (In""t Veld et al, Diabetologia 35:272-276,1992); in the human pancreas, they remain numerous throughout adult life (Bouwens and Pipeleers, Diabetologia 41:629-633, 1993). Since the migration and association of pancreatic endocrine cells into typical islet structures is considered as a step in maturation (Pictet and Rutter, development of the embryonic pancreas. In: Steiner D F, Freinkel N (eds) Handbook of Physiology, Section 7 Endocrinology Vol I: Endocrine Pancreas, Baltimore; Williams and Wilkins, 1972 pp25-66), the endocrine cells which do not occur in these micro-organs can be defined as xe2x80x9cimmaturexe2x80x9d. Although the properties of immature beta cells and immature endocrine cells have not been well characterized, they are likely different from those of the xe2x80x9cisletsxe2x80x9d which have matured under influence of their typical microanatomy and neighbouring endocrine cells (Orci and Unger, The Lancet 2;1243-1244,1975; Pipeleers, Experientia 440:1114-1126,1984; Pipeleers, Diabetologia 30:277-291, 1987). The islet functions are considered as typical for mature beta cells; mature beta cells are larger than their immature counterparts (Pipeleers, unpublished observations). There is indirect evidence that the xe2x80x9cimmaturexe2x80x9d beta cells can achieve a growth of the beta cell mass. The loss of these cells during the isolation procedure is thus expected to result in a purified endocrine cell preparation which is only representative for the mature cell population, which contains only a subpopulation of the beta cells and which exhibits a low capacity for growth, three consequences that are disadvantageous when the isolated cells are to be used for the above-mentioned strategies, namely the construction of beta cell grafts and the development of drugs which aim to increase the functional beta cell mass.
In order to overcome said problems the invention provides a method according to claim 1. Preferred embodiments of the method according to the invention are described in subclaims 2 to 18. The preparations per se and heir use are subject of claims 19-22.
In the broadest aspect of the invention a method for the preparation of mammalian pancreatic endocrine cells is provided comprising the steps of dissociating intact pancreatic tissue into a cell suspension containing single cells and of enriching said cell suspension in immature and/or mature endocrine cells.
Intact pancreatic tissue is defined herein as the pancreatic organ or any of its segments after its dissection from the mammalian body.
A pancreatic endocrine cell is defined as a cell which is found in, or is isolated from, intact pancreatic issue and which expresses an endocrine marker, i.e. a molecule that has been identified in endocrine but not in exocrine cells. This marker can correspond to a constituent of an endocrine secretary vesicle or any other cell component.
Immature endocrine cells are defined by one or more of the following criteria: 1) a cellular phenotype that is characteristic for fetal endocrine cells but not for adult endocrine cells, for example the presence of gastrin immunoreactivity, or of synaptophysin immunoreactivity, without a positivity for any of the adult pancreatic hormones i.e. insulin, glucagon, somatostatin, pancreatic polypeptide. 2) occurrence in pancreatic tissue as unit of maximally four endocrine cells, 3) expression of a marker which is not found in homologous endocrine cells of adult pancreatic islets, such as cytokeratin 19 (CK19; human) or cytokeratin 7 (CK7; pig). Immature beta cells characteristically present a significantly smaller cell size when compared to adult beta cells (smaller than the mean diameter of adult beta cells minus three standard deviations) and a poor responsiveness to a maximal glucose stimulus ( less than 3 fold stimulation of insulin release); the size these cells increases under maturation conditions.
Mature endocrine cells are defined by their location in pancreatic islets which are known to exhibit a typical vascularization (Bonner-Weir and Orci, Diabetes 31, 883-889, 1982; Pictet and Rutter, In: Steiner D F, Freinkel N (eds) Handbook of Physiology, Section 7 Endocrinology Vol I: Endocrine Pancreas, Baltimore: Williams and Wilkins, 1972 pp25-66).
Mature beta cells are characterized by their glucose-regulated biosynthesis and release of insulin, and their storage and release of insulin that is  greater than 90 percent processed into its mature form.
In contrast to previously described, and routinely used, methods for the isolation of pancreatic islets (Lacy P E and Kostianovsky M, Diabetes 16:35-39, 1967) the invention does not intend to isolate the xe2x80x9cislets of Langerhansxe2x80x9d under the form at which they occur in the intact pancreas. Instead, the whole pancreatic organ, including the xe2x80x9cislets of Langerhansxe2x80x9d is dissociated into single cells and small cellular aggregates before steps are taken to isolate the mature and immature endocrine cells.
By selecting the age of the pancreas, and the experimental conditions, the method can preferentially yield mature or immature endocrine cells or their subtypes. Thus, dissociation of late-fetal pig pancreata, elutriation of single cells from this dissociate, and culture of the elutriation fraction under specific conditions allows a large-scale purification of immature endocrine cells, both of the beta and alpha cell types. The invention describes the specific conditions under which this preparation can be used 1) to produce grafts with an important potential of beta cell growth in vivo, 2) to design and perform drug screening tests in vitro. In general, it provides the method for producingxe2x80x94from pancreata of different ages and speciesxe2x80x94endocrine cell preparations of selected cellular composition and properties for use as auto-, allo- and xenografts as well as for screening and assessing drugs in the laboratory.
This invention describes the production if preparations of mature and immature endocrine cells from the mammalian pancreas for use in the treatment of diabetes, more specifically the production of beta cell grafts with a growth potential, and the provision of experimental models in which drugs can be screened for their therapeutic effects on the functional beta cell mass.
The method can be used for a large scale production of endocrine cells from pancreata of various species, especially from late-gestation fetal pancreata and more specifically from fetal porcine pancreas, yielding preparations of defined size and cellular composition, selected purity in beta and alpha cells, predictable insulin biosynthetic capacity and ability of beta cell growth.
This particular application using late fetal porcine pancreas offers the following advantages in comparison to postnatal preparations from large species:
1. a lower risk of infection
2. higher purity in endocrine cells
3. larger endocrine cell number per technical procedure
4. distinct capacity for growth and longer survival of the beta cell mass
5. more reproducible functional properties
In comparison to other methods for the isolation of fetal endocrine cells the method offers the following advantages:
1. higher purity in endocrine cells and their subtypes
2. larger endocrine cell number per organ
3. ability to select particular endocrine cell (sub) types and compose final preparations according to the metabolic needs.
4. availability of immature endocrine cells
These properties make the method useful for 1) the preparation of cell grafts for transplantation in diabetic patients 2) the preparation of cell preparations or screening of drugs which regulate the functional beta cell mass.
The most preferred embodiment of the method comprises six steps which, in combination, yield pancreatic endocrine cell preparations of selected size, composition and maturity;
1. dissociation to intact pancreatic tissue to the point where all tissue, including the islets of Langerhans, are preferably dispersed into cellular aggregates of  less than 100 xcexcm.
2. separation of the pancreatic dissociate according to particle size using counterflow centrifugation or elutriation to select single cells with size 6 to 15 xcexcm (including immature endocrine cells) and small aggregates with size 15 to 100 xcexcm (including mature endocrine cells).
3. elimination of non-endocrine or acinar cells from selected fractions by density gradient centrifugation.
4. enrichment in mature or immature endocrine cells by culture in specially formulated serum-free media.
5. purification of mature or immature endocrine, alpha or beta cells and their precursors by fluorescence-activated cell sorting.
6. composition of endocrine cell preparations with selected size, composition and maturity.
Steps 1 through 4 are used to purity mature and immature endocrine cells from 1 to 5 percent in intact tissue to a minimum of 60 percent and preferably 90 percent. Step 5 is necessary if a further purification is needed into (im) mature endocrine, alpha or beta cell enriched preparations. This procedure allows a large scale isolation of pancreatic endocrine cells while offering the possibility of selecting cells according to the experimental needs. A higher purity in intact endocrine cells is associated with a lower immunogenicity (Pipeleers et al, Diabetes 40, 920-930, 3991; Pipeleers-Marichal et al, Diabetes 40, 931-938, 1991; Pipeleers et al, Diabetologia 34; 390-396, 1991), inclusion of immature endocrine or beta cells is associated with a higher growth potential, addition of alpha cells increases and promotes the survival and function of beta cells (Pipeleers, Diabetologia 30, 277-291, 1987; Ling et al, Diabetologia 37, 15-21, 1994; Keymeulen et al, Diabetologia 40:1152-1158, 1997), standardized beta cell preparations are needed for standardized metabolic effects in diabetes (Keymeulen et al, Diabetologia 41:452-459, 1998), purified cell populations are required for drug testing (Pipeleers et al, Endocrinology 117:806-816, 1985; Gorus et al, Diabetes 37, 1090-1095, 1988).
Step 1
The preparation of pancreatic endocrine tissue is classically performed by collagenase digestion of the pancreatic gland and by separating a fraction enriched in islets of Langerhans using methods which isolate larger ( greater than 100 xcexcm) tissue particles (by manual isolation under the dissection microscope) or particles with lower density ( less than 1.07 g/ml). These fractions are enriched in islets of Langerhans and can be used to purify endocrine beta cells and alpha cells (Pipeleers et al, Endocrinology 117:806-816, 1985). These methods have the disadvantage that they result in the loss of (immature) endocrine cells that are contained in smaller tissue particles or/and in particles with higher density. This loss can be significant in quantitative and in qualitative terms as it will mean the loss of (immature) cells and small cell aggregates which are important for the growth of the beta cell mass. We have therefore chosen another methodologic strategy which starts with the dissociation of intact pancreatic tissue to the point, that all tissue, including the islets of Langerhans, is disassembled into particles  less than 100 xcexcm. These particles then form the basis for all subsequent processing steps.
Use of this stategy allows the isolation of larger numbers of endocrine cells, including immature endocrine and immature beta cells, than the classical. xe2x80x98islet of Langerhansxe2x80x99 based approach.
In step 1, the pancreatic tissue is mechanically dispersed during and following an incubation, first with collagenase for maximally 30 minutes and than with a calcium chelator EDTA (or EGTA) in a calcium-free medium containing DNase for maximally 30 minutes. The collagenase concentrations are determined per enzyme batch and set on the basis of, respectively, a disassembling of the intact tissue into particles with diameter  less than 500, within 20 minutes, and maintaining this preparation free of strands and subsequent cell clumping. The calcium-free medium reduces cell adhesion and allows gentle dissociation of the endocrine tissue into single cells and small cellular aggregates. Immature endocrine cells will be present in particles smaller than 100 xcexcm, and, in fetal pancreata, particularly as single cells with diameter 6-15 xcexcm. Most mature endocrine cells will occur in particles with diameter 15-100 xcexcm. Particles larger than 100 xcexcm are removed by a 100 xcexcm screen or by counterflow elutriation in which the smaller particles are collected at a flow rate of 225 ml/min and a rotor speed of 250 rpm (Beckman centrifuge J-6B, rotor JE10x).
Step 2
In this step single immature endocrine cells are separated from aggregated cells, including mature endocrine cells, while discarding debris. It is based on our finding that application of step 1 on fetal tissue results in release of immature endocrine cells as single units with a diameter of 6-15 xcexcm. It is achieved by our adaptation of the technique of counterflow elutriation as to separate particles with diameter 6-15 xcexcm from those with diameter 15-100 xcexcm. The particle suspension is pumped at 25 ml/min into a Beckman (Palo Alto, Calif.) JE10x elutriator placed in a Beckman J6B centrifuge at 1500 rpm, whereby debris will be flushed cut and particles  greater than 6 xcexcm are retained in the chamber. The pump speed will hen be increased to 190 ml/min in order to push the particles with diameter 6-15 xcexcm out of the rotor chamber: this fraction is collected ( less than 15 xcexcm fraction) and contains the single immature endocrine cells. The speed of the centrifuge is then reduced to 0 rpm so that the content of the rotor can be collected as the 15-100 xcexcm fraction. When applied on the  less than 100 xcexcm fraction of a dissociate from late-gestation fetal pig pancreata, all endocrine cells in the  less than 15 xcexcm fraction exhibit markers of immaturity (lack of typical glucose responsiveness, expression of duct cell marker CK7).
Step 3
The two fractions that are isolated after counterflow elutriation are contaminated by non-endocrine or acinar cells which exhibit a higher density ( greater than 1.070 g/ml) than endocrine cells. Density gradient centrifugation is used to reduce the level of contamination: the cell fractions from late-gestation fetal porcine pancreata are submitted to discontinuous Percoll gradients with densities of 1.040 g/ml and 1.075 g/ml. Endocrine-cell enriched preparations are collected at the interphase of density layers 1.40 and 1.075 g/ml; they yield preparations with  greater than 40 percent intact endocrine cells. The endocrine cells in the interphase from the 6-15 xcexcm fraction are all immature; their number is consistently between 1 and 3 107 cells per fetal (porcine) pancreas.
Considering the mean number of fetuses (n=9) per sowxe2x80x94and hence per isolation experimentxe2x80x94, the yield in endocrine cells is minimally 108 endocrine cells per isolation at a purity of  greater than 40%. The procedure therefore offers a large scale isolation of endocrine cells. This yield is higher than that from a human donor pancreas; it is also reproduced consistently.
At the end of step 3, the interphases also contain a proportion of non-granulated cells several of which are attached to endocrine cells. This non-granulated cell populationxe2x80x94or a fraction thereoffxe2x80x94is to contributes to the growth of the beta cell mass through immature endocrine cells, i.e. cells which can which can differentiate into beta cells.
Step 4
The two fractions collected from step 3, namely interphase of  less than 15 xcexcm and interphase 15-100 xcexcm, are further enriched in immature and mature endocrine cells by culture in specially formulated media:
for immature endocrine cells this medium is serum-free Ham""s F10 with albumin (max 0.5%) as protein and supplements of nicotinamide (5 mM), glucocorticoids (max 10xe2x88x926 M hydrocortisone), isobutylmethylxanthine (IBMX, 50 xcexcM); compared to the media used for culture of islets of Langerhans this medium preserves the survival of immature endocrine cells and allows their increased storage of hormone during prolonged culture (2-fold increase in cellular insulin content after 4 weeks of culture); The degree of maturation and differentiation is suppressed by addition of serum and increasing the concentration of calcium to 2 mM; these latter supplements can be used to prepare fractions with more beta cell precursor cells.
for mature endocrine cells the culture medium is similar to that previously described for human beta cells (Ling and Pipeleers, J Clin Invest 98, 2805-2812, 1996), namely serum-free Ham""s F10 with albumin (0.5%) and IBMX (50 xcexcM).
After four days culture of the interphase  less than 15 xcexcm, the proportion of damaged cells is reproducibly under 10 percent, of intact endocrine cells above 70 percent, of non-granulated cells between 10 and 25 percent. The beta cells synthesize proinsulin at a rate of minimally 10 fmol/103 beta cells/hour. These quality control tests for cellular composition and functions are extended with microbiologic and toxicologic control tests.
For human implantation purposes the source animals are deprived from herds that are specific pathogen free according to the norms of the Federation of European Laboratory Animal Science Associations (FELASA) as defined this association (Laboratory Animals 32:1-17, 1998) and that comply with biological safety standards as proposed by the US Food and Drug Administration (Federal Register 49920-49932, August 1996).
The cell preparations described above can be used for transplantation and correction of diabetes in mice; they were shown to achieve growth of their beta cell mass (see example V). They can be used to purify composing cell (sub) types (step 5) and to design experimental models of single and aggregated cells with selected size, composition and maturity (step 6).
Step 5
The preparations obtained from step 4 can be dissociated into single cells using trypsin and DNase. The cell suspension is then submitted to fluorescence-activated cell sorting (FACS) using forward scatter (FSC), sidewards scatter (SSC) and fluorescence (FL) at 488 nm excitation and 520-540 nm emission as discrimination parameters. Populations of cell (sub) types are distinguished with respect to the location of intact non-granulated cells including immature endocrine cells (low FSC, low SSC, low FL); at higher SSC: enrichment in alpha cells; at higher FSC and FL: enrichment in beta cells whereby beta cell precursors and immature beta cells exhibit lower FSC and FL than mature beta cells.
Windows are set for the purification of these cell (sub) types as single cells.
Step 6
The cell (sub) types collected after step 5 can be cultured as single cells on polylysine-coated culture plates using the media defined in step 4 for immature or mature endocrine cells. The non-granulated cells with the immature endocrine cells (containing beta cell precursors) are cultured in the medium for immature endocrine cells 10 percent fetal serum.
The cells described under steps 4 and 5 can also be reaggregated into particles of selected size by gyratory shaking incubation in a CO2 incubator using the media specified in the preceding paragraph and at cell densities from 104 to 2.105 per cm2 surface of the bacteriologic dishes used for suspension culture. Cell aggregates of increasing size are obtained by increasing the cell density and increasing the speed and duration of gyratory shaking. Aggregates of varying composition can also be formed by mixing purified cell populations in varying proportions.
The preparations obtained by step 4 and 5 can be used for transplantation and for short- or long-term culture. They are useful in the treatment of diabetes, namely as a source of insulin in diabetic recipients and as an in vitro or in vivo model for drug screening.
The in vitro composed grafts and the cultured cell preparations can be submitted to functional analysis as well as to the microbiologic quality control tests that are required by regulatory authorities. The cell preparations can thus be screened for safety and efficacy while kept in culture and before actual implantation in diabetic patients.